1. Field of the Invention
The present invention relates to an optical system in a confocal optical scanner, and particularly to improvements in a dichroic mirror for measuring a plurality of kinds of fluorescence (also referred to as a polychromatic fluorescence) emitted from a sample at the same time and enhancement of the fluorescence efficiency.
2. Description of the Related Art
Conventionally, a confocal optical scanner used with a microscope has been well known (e.g., refer to C. Genka, K. Ishida, K. Ichimori, Y. Hirota, T. Tanaami, H. Nakazawa, “Visualization of biphasic Ca2+ diffusion from cytosol to nucleus in contracting adult rat cardiac myocytes with an ultra-fast confocal imaging system”, Cell Calcium, Volume 25, Issue 3, P. 199-208). FIG. 1 is a schematic diagram of the principle of the confocal optical scanner of this kind. A laser 1 as an excited light is converged into individual light fluxes by each microlens 3 disposed on a microlens disk 2, transmitted through a multi-chroic mirror (hereinafter referred to as a multi-wavelength dichroic mirror, though it should be essentially called multi-chroic mirror) 4, and then passed through individual pinholes 6 provided on a pinhole disk (also called a Nipkow disk) 5 to be converged by an objective lens 7 onto a sample 8.
Fluorescence emitted from the sample 8 is passed through the objective lens 7 again and converged into individual pinholes on the pinhole disk 5. Fluorescence passed through individual pinholes 6 is reflected by the multi-wavelength dichroic mirror 4, and passed through a relay lens 9 to form a fluorescent image on a sensor 10.
The multi-wavelength dichroic mirror 4 used herein is designed to pass excited light 1 and to reflect fluorescence from the sample 8.
The microlens disk 2 and the pinhole disk 5 are mechanically coupled through a member 11, and integrally rotate around an axis of rotation 12. Individual microlenses 3 and pinholes 6 are disposed so that excited light from individual pinholes 6 formed on the pinhole disk 5 scans over an observation plane of the sample 8. Since a plane where the pinholes 6 are arranged, the observation plane of the sample 8, and a light receiving plane of the sensor 10 are respectively disposed in optically conjugate relation, an optically sectional image of the sample 8, that is, a confocal image is imaged on the sensor 10.
In such a confocal optical scanner, the multi-wavelength dichroic mirror 4 for measuring the multiple wavelengths at the same time is employed. The characteristic of the multi-wavelength dichroic mirror 4 is as shown in FIG. 2A, for example. The corresponding characteristic, that is, the wavelength characteristic of fluorescence emitted from the sample 8 is as shown in FIG. 2B.
In the conventional confocal optical scanner, the multi-wavelength dichroic mirror 4 is employed. The multi-wavelength dichroic mirror 4 passes the excited light of a wavelength band with low reflectance, and reflects fluorescence from the sample 8 of a wavelength band with high reflectance to lead it to the sensor 10.
When a single wavelength dichroic mirror is employed, it is required that three dichroic mirrors having different characteristics are mechanically switched. However, when the multi-wavelength dichroic mirror is employed, there is the advantage that polychromatic fluorescent images can be measured at the same time without exchanging and switching the dichroic mirrors.
However, the conventional confocal optical scanner has the following problems.    (1) This multi-wavelength dichroic mirror cannot reflect a considerable part of proper fluorescence emitted from the sample.
For example, cyan fluorescent labeled protein ECFP, yellow fluorescent labeled protein EYFP, red fluorescent protein DsRed are considered regarding the expression vector as follows.
(a) For ECFP, about 60% on the side of the longer wavelength than 480 nm is cut.
(b) For EYFP, about 30% on the side of the longer wavelength than 560 nm is cut.
(c) For DsRed, about 60% on the side of the shorter wavelength than 600 nm including peaks is cut.
The efficiency is very bad with the characteristic of such a multi-wavelength dichroic mirror, so that the obtained fluorescent image is dark, and the S/N ratio is low.    (2) A dedicated filter is required for each kind of fluorescent reagent.
As shown in FIG. 2B, the wavelength characteristic of fluorescent reagent vary from one reagent to another. Further, the corresponding multi-wavelength dichroic mirror must be designed and prepared for each combination thereof. It is undesirable in respects of cost, inventory, exchange time and contamination at exchange time. Also, when the filter is exchanged, a large image deviation may occur as a significant problem when the superposed image is observed.    (3) When the transmitted image or the inverted fluorescent image of blue nucleus staining with 4′,6-diamidino-2-phenylindole, dihydrochioride (Dapi) color pigment is superposed on the confocal fluorescent image to be measured, a blue image, as indicated by the one dot chain line in FIG. 2A, of the transmitted image or the inverted fluorescent image is cut by the multi-wavelength dichroic mirror. Therefore, the correct measurement is not allowed, and the superposed image is dark.